XL Agar Base

References Availability 1. Wilkins and Chalgren. 1976. Antimicrob. Agents Chemother. 10:926. Difco™ Wilkins-Chalgren Agar 2. National Committee for Clinical Laboratory Standards. 1993. Methods for antimicrobial suscepti- bility testing of anaerobic bacteria. Approved standard M11-A3. NCCLS, Villanova, Pa. NCCLS 3. Wexler and Doern. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of clinical , 6th ed. American Society for Microbiology, Washington, D.C. Cat. No. 218051 Dehydrated – 500 g 4. Isenberg (ed.). 1995. Clinical microbiology procedures handbook, vol 1. American Society for ™ Microbiology, Washington, D.C. Difco Anaerobe Broth MIC NCCLS Cat. No. 218151 Dehydrated – 500 g

XL Agar Base • XLD Agar

Intended Use It is particularly recommended for obtaining counts of enteric XLD Agar conforms with specifications of The United States organisms. This medium can be rendered moderately selective Pharmacopeia (USP). for enteric pathogens, particularly , by the addition of sodium desoxycholate (2.5 g/L) to make XLD Agar.1 XL (Xylose Lysine) Agar Base is used for the isolation and differ- entiation of enteric pathogens and, when supplemented with XL Agar Base can be made selective for by adding appropriate additives, as a base for selective enteric media. 1.25 mL/L of 1% aqueous brilliant green to the base prior to autoclaving. Its use is recommended for Salmonella isolation XLD Agar is the complete Xylose Lysine Desoxycholate Agar, after selenite or tetrathionate enrichment in food analysis; both a moderately selective medium recommended for isolation and coliforms and Shigella are inhibited.1 differentiation of enteric pathogens, especially Shigella species. XLD Agar was developed by Taylor in order to increase the Summary and Explanation efficiency of the isolation and identification of enteric pathogens, A wide variety of media have been developed to aid in the particularly Shigella.1 The pathogens are differentiated not selective isolation and differentiation of enteric pathogens. Due only from the nonpathogenic lactose fermenters but also from to the large numbers of different microbial species and strains many nonpathogens which do not ferment lactose or sucrose. with varying nutritional requirements and chemical resistance Additionally, the medium was formulated to increase the patterns, investigators have developed various formulae to meet frequency of growth of the more fastidious pathogens,1 which general as well as specific needs relative to isolation and in other formulations have often failed to grow due to the identification of the microorganisms. inclusion of excessively toxic inhibitors. The results obtained in a number of clinical evaluations have supported XL Agar Base was developed by Taylor1 for the nonselective the claim for the relatively high efficiency of XLD Agar in the isolation and differentiation of gram-negative enteric bacilli. XL Agar Base XLD Agar U Uninoculated Escherichia coli Salmonella typhimurium Shigella flexneri ™ ™ ™ - Plate ATCC 25922 ATCC 14028 ATCC 12022 Z

625 Section III U-Z XL Agar Base, cont.

User Quality Control NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications Difco™ XLD Agar Dehydrated Appearance: Pink, free-flowing, homogeneous. Solution: 5.7% solution, soluble in purified water upon boiling. Solution is red, very slightly to slightly opalescent. Prepared Appearance: Red, slightly opalescent. Reaction of 5.7% Solution at 25°C: pH 7.4 ± 0.2

Cultural Response Difco™ XLD Agar Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours. INOCULUM ORGANISM ATCC™ CFU RECOVERY COLONY COLOR faecalis 29212 103-2×103 Partial inhibition – Escherichia coli 25922 103-2×103 Partial inhibition Yellow with or without bile precipitate Providencia alcalifaciens 9886 102-103 Good Red Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium 14028 102-103 Good Red with black centers Shigella flexneri 12022 102-103 Good Red Continued

primary isolation of Shigella and Salmonella.2-6 XLD Agar Formulae is included in the USP microbial limit test for screening specimens BBL™ XL Agar Base for the presence or absence of Salmonella7 and is recommended Approximate Formula* Per Liter for the testing of foods, dairy products and water.7-12 Xylose ...... 3.5 g L-Lysine ...... 5.0 g Lactose ...... 7.5 g Principles of the Procedure Sucrose ...... 7.5 g Xylose is incorporated into the medium since it is fermented Sodium Chloride ...... 5.0 g Yeast Extract ...... 3.0 g by practically all enterics except for the shigellae, and this prop- Phenol Red ...... 0.08 g erty enables the differentiation of Shigella species. Lysine is Agar ...... 13.5 g included to enable the Salmonella group to be differentiated Difco™ XLD Agar from the nonpathogens since, without lysine, salmonellae Approximate Formula* Per Liter rapidly would ferment the xylose and be indistinguishable from Xylose ...... 3.75 g nonpathogenic species. After the salmonellae exhaust the L-Lysine ...... 5.0 g Lactose ...... 7.5 g supply of xylose, the lysine is attacked via the enzyme, lysine Saccharose ...... 7.5 g decarboxylase, with reversion to an alkaline pH which mimics Sodium Chloride ...... 5.0 g the Shigella reaction. To prevent similar reversion by lysine- Yeast Extract ...... 3.0 g Phenol Red ...... 0.08 g positive coliforms, lactose and sucrose (saccharose) were added Sodium Desoxycholate ...... 2.5 g to produce acid in excess.1 Sodium Thiosulfate ...... 6.8 g Ferric Ammonium Citrate ...... 0.8 g To add to the differentiating ability of the formulation, an H2S Agar ...... 15.0 g indicator system, consisting of sodium thiosulfate and ferric BBL™ XLD Agar ammonium citrate, is included for the visualization of the Approximate Formula* Per Liter hydrogen sulfide produced, resulting in the formation of colonies Xylose ...... 3.5 g L-Lysine ...... 5.0 g with black centers. The nonpathogenic H2S producers do not Lactose ...... 7.5 g decarboxylate lysine; therefore, the acid reaction produced by Sucrose ...... 7.5 g them prevents the blackening of the colonies.1 Sodium Chloride ...... 5.0 g Yeast Extract ...... 3.0 g XLD Agar is both a selective and differential medium. It utilizes Phenol Red ...... 0.08 g sodium desoxycholate as the selective agent and, therefore, it Sodium Desoxycholate ...... 2.5 g Sodium Thiosulfate ...... 6.8 g is inhibitory to gram-positive microorganisms. Ferric Ammonium Citrate ...... 0.8 g Agar ...... 13.5 g *Adjusted and/or supplemented as required to meet performance criteria.

626 XL Agar Base, cont.

Identity Specifications BBL™ XL Agar Base Dehydrated Appearance: Fine, homogeneous, free of extraneous material. Solution: 4.5% solution, soluble in purified water upon boiling. Solution is dark medium to dark, red to rose-red, clear to slightly hazy. Prepared Appearance: Dark medium to dark, red to rose-red, clear to slightly hazy. Reaction of 4.5% Solution at 25°C: pH 7.5 ± 0.2 BBL™ XLD Agar Dehydrated Appearance: Fine, homogeneous, free of extraneous material. Solution: 5.5% solution, soluble in purified water upon boiling. Solution is medium to dark, orange-red to rose-red, clear to slightly hazy. Prepared Appearance: Medium to dark, orange-red to rose-red, clear to slightly hazy. Reaction of 5.5% Solution at 25°C: pH 7.4 ± 0.2

Cultural Response BBL™ XL Agar Base Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours (up to 48 hours if necessary). ORGANISM ATCC™ INOCULUM CFU RECOVERY COLONY COLOR Escherichia coli 25922 103-104 Good Yellow without precipitate Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium 14028 103-104 Good Red to yellow without precipitate, black centers to predominately black Shigella flexneri 12022 103-104 Good Red BBL™ XLD Agar Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours.

ORGANISM ATCC™ INOCULUM CFU RECOVERY COLONY COLOR Enterococcus faecalis 29212 104-105 Partial to complete inhibition – Escherichia coli 25922 104-105 Partial to complete inhibition If growth, yellow with or without bile precipitate Klebsiella pneumoniae 33495 103-104 Good Yellow with or without bile precipitate Proteus vulgaris 8427 103-104 Good Yellow with or without bile precipitate 10145 103-104 Good Red Salmonella choleraesuis U subsp. choleraesuis serotype Typhi 19430 103-104 Good Red with black centers - Salmonella choleraesuis Z subsp. choleraesuis serotype Paratyphi 9150 103-104 Good Red Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium 14028 103-104 Good Red with black centers Shigella boydii 9207 103-104 Good Red Shigella flexneri 12022 103-104 Good Red

Directions for Preparation from 5. Test samples of the finished product for performance using Dehydrated Product stable, typical control cultures. BBL™ XL Agar Base Difco™ and BBL™ XLD Agar 1. Suspend 45 g of the powder in 1 L of purified water. Mix 1. Suspend the powder in 1 L of purified water: thoroughly. Difco™ XLD Agar – 57 g; 2. Heat with frequent agitation and boil for 1 minute to com- BBL™ XLD Agar – 55 g. pletely dissolve the powder. Add brilliant green, if desired. Mix thoroughly. 3. Autoclave at 118°C for 10 minutes. Cool to 55-60°C. 2. Heat with agitation just until the medium boils. DO NOT 4. Add 20 mL of an aqueous solution containing 34% OVERHEAT. DO NOT AUTOCLAVE. sodium thiosulfate and 4% ferric ammonium citrate. 3. Cool to 45-50°C in a water bath and use immediately. For XLD agar, add 25 mL of 10% aqueous sodium Overheating causes precipitation. desoxycholate. Pour into plates. 4. Test samples of the finished product for performance using stable, typical control cultures. 627 Section III U-Z XL Agar Base, cont.

Procedure References 1. Taylor. 1965. Am. J. Clin. Pathol. 44:471. Use standard procedures to obtain isolated colonies from 2. Taylor and Harris. 1965. Am. J. Clin. Pathol. 44:476. specimens. A nonselective medium should also be streaked to 3. Taylor and Harris. 1967. Am. J. Clin. Pathol. 48:350. 4. Taylor and Schelhart. 1967. Am. J. Clin. Pathol. 48:356. increase the chance of recovery when the population of gram- 5. Taylor and Schelhart. 1968. Appl. Microbiol. 16:1387. 6. Pollock and Dahlgren. 1974. Appl. Microbiol. 27:197. negative organisms is low and to provide an indication of other 7. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The national formulary 20 – 2002. United States Pharmacopeial Convention, Inc., Rockville, Md. organisms present in the specimen. Incubate plates, protected 8. U.S. Food and Drug Adminsitration. 1995. Bacteriological analytical manual, 8th ed. AOAC ± ° International, Gaithersburg, Md. from light, at 35 2 C for 18-24 hours. Colonies on XLD agar 9. Horwitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed. AOAC Interna- may require 48 hours incubation for full color development. tional, Gaithersburg, Md. 10. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. 11. Marshall (ed.). 1993. Standard methods for the examination of dairy products, 16th ed. American Expected Results Public Health Association, Washington, D.C. 12. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and Degradation of xylose, lactose and sucrose generates acid prod- wastewater, 20th ed. American Public Health Association, Washington, D.C. ucts, causing a color change in the medium from red to yellow. Hydrogen sulfide production under alkaline conditions causes Availability ™ colonies to develop black centers. This reaction is inhibited by BBL XL Agar Base the acid conditions that accompany carbohydrate fermentation. SMWW Cat. No. 211836 Dehydrated – 500 g Lysine decarboxylation in the absence of lactose and sucrose Difco™ XLD Agar fermentation causes reversion to an alkaline condition and the AOAC BAM BS10 CMPH COMPF EP MCM7 SMD color of the medium changes back to red. SMWW USP Cat. No. 278850 Dehydrated – 500 g Typical colonial morphology and reactions on XLD Agar are 278820 Dehydrated – 2 kg as follows: 278830 Dehydrated – 10 kg ™ E.coli ...... Large, flat, yellow; some strains BBL XLD Agar may be inhibited AOAC BAM BS10 CMPH COMPF EP MCM7 SMD Enterobacter/ Klebsiella ...... Mucoid, yellow SMWW USP Proteus ...... Red to yellow; most strains have Cat. No. 211838 Dehydrated – 500 g black centers 212266 Dehydrated – 5 lb (2.3 kg) Salmonella ...... Red-yellow with black centers United States and Canada Shigella, Salmonella Cat. No. 221192 Prepared Plates – Pkg. of 20* H S-negative ...... Red 2 221284 Prepared Plates – Ctn. of 100* Pseudomonas ...... Red Gram-positive bacteria ...... No growth to slight growth Europe Cat. No. 254055 Prepared Plates – Pkg. of 20* Limitations of the Procedure 254090 Prepared Plates – Ctn. of 120* Japan 1. Red, false-positive colonies may occur with some Proteus Cat. No. 251159 Prepared Plates – Ctn. of 100* and Pseudomonas species. BBL™ XLD Agar// 2. Incubation in excess of 48 hours may lead to false-positive Cat. No. 295646 Prepared I Plate™ Dishes – Pkg. of 20* results. *Store at 2-8°C. 3. S. paratyphi A, S. choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species. 4. Some Proteus strains will give black-centered colonies on XLD Agar.

XLT4 Agar Base • XLT4 Agar Supplement

Intended Use isolation media where overgrowth of Proteus, Providencia and XLT4 Agar Base is used with XLT4 Agar Supplement in Pseudomonas can dramatically interfere with the detection and isolating non-typhi Salmonella. isolation of Salmonella. In 1990, Miller and Tate described a new medium, XLT4 Agar, Summary and Explanation for isolating Salmonella.1 The authors established the selectivity Numerous media have been developed for isolating and differ- of XLT4 Agar using pure cultures of a variety of enteric organ- entiating enteric pathogens. The majority were designed to isms. They also evaluated its sensitivity in detecting and isolating 1 recover a broad spectrum of enteric pathogens. Consequently, Salmonella using fecal-contaminated farm samples containing high overgrowth of nuisance or contaminating organisms can be numbers of competing bacteria. In follow-up studies, Miller2,3 a major problem when recovery of a specific organism or and Tate4 reported that XLT4 Agar significantly improved the species is desired. This is particularly true for Salmonella

628 XLT4 Agar Base, cont.

User Quality Control XLT4 Agar Base with XLT4 Supplement Salmonella typhimurium Identity Specifications ATCC™ 14028 Difco™ XLT4 Agar Base Dehydrated Appearance: Pink, free flowing, homogeneous. Solution: 5.9% solution, soluble upon boiling in purified water containing 4.6 mL/L of XLT4 Agar Supplement. Solution is red, slightly opalescent. Prepared Appearance: Reddish-orange, slightly opalescent. Reaction of Final Medium at 25°C: pH 7.4 ± 0.2 Difco™ XLT4 Agar Supplement Appearance: Colorless to slightly yellow, clear, slightly viscous solution.

Cultural Response Difco™ XLT4 Agar Base with XLT4 Agar Supplement Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours. INOCULUM COLONY ORGANISM ATCC™ CFU RECOVERY COLOR Enterococcus faecalis 29212 103 Marked inhibition – Escherichia coli 25922 103 Partial inhibition Yellow Proteus mirabilis 25933 103 Inhibition – Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium 14028 102-103 Good Yellow to red with black centers Staphylococcus aureus 25923 103 Inhibition – recovery of non-typhi Salmonella from chicken and farm Difco™ XLT4 Agar Supplement environmental drag-swab samples. A 27% solution (approximate) of the surfactant Tergitol™** 4 (7-ethyl-2-methyl-4-undecanol hydrogen sulfate, sodium salt). *Adjusted and/or supplemented as required to meet performance criteria. Principles of the Procedure **Tergitol is a trademark of Union Carbide Chemicals & Plastics Technology Corporation. XLT4 Agar Base contains peptone as a source of complex Directions for Preparation from nitrogen compounds. Yeast extract is added as a source of U vitamins and other cofactors. Differentiation of Salmonella Dehydrated Product - from other organisms that also grow on this medium is based 1. Suspend 59 g of the powder in 1 L of purified water. on fermentation of xylose, lactose and sucrose, decarboxylation 2. Add 4.6 mL XLT4 Agar Supplement. Mix thoroughly. Z of lysine and the production of hydrogen sulfide. Hydrogen 3. Heat with frequent agitation and boil for 1 minute to com- sulfide production is detected by the addition of ferric ions. pletely dissolve the powder. Avoid overheating. DO NOT Sodium thiosulfate is added as a source of inorganic sulfur. AUTOCLAVE. Sodium chloride maintains the osmotic balance of the medium. 4. Test samples of the finished product for performance using Agar is the solidifying agent. Phenol red is added as an indicator stable, typical control cultures. of pH changes resulting from fermentation and decarboxylation reactions. XLT4 Agar Supplement is added to inhibit growth Procedure of non-Salmonella organisms. 1. Inoculate a suitable Salmonella enrichment broth (such as Tetrithionate Broth) and incubate at 35°C for 18-24 hours. Formulae 2. Following enrichment, subculture onto XLT4 Agar. Streak Difco™ XLT4 Agar Base for isolation. Approximate Formula* Per Liter 3. Incubate plates aerobically at 35 ± 2°C. Examine for growth Proteose Peptone No. 3 ...... 1.6 g after 18-24 and 48 hours incubation. Yeast Extract ...... 3.0 g L-Lysine ...... 5.0 g Xylose ...... 3.75 g Expected Results Lactose ...... 7.5 g Typical Salmonella colonies (H2S-positive) appear black or Saccharose ...... 7.5 g Ferric Ammonium Citrate ...... 0.8 g black-centered with a yellow periphery after 18-24 hours of Sodium Thiosulfate ...... 6.8 g incubation. Upon continued incubation, the colonies become Sodium Chloride ...... 5.0 g entirely black or pink to red with black centers. Agar ...... 18.0 g Phenol Red ...... 0.08 g 629 Section III U-Z XLT4 Agar Base, cont.

Colonies of H2S-negative Salmonella strains appear pinkish- 3. Freshly inoculated plates and plates held over several days yellow. may develop multicolored, metallic looking crystals/flecks on the surface. These crystals/flecks do not interfere with Most Citrobacter colonies that grow on this medium are yellow the performance of the medium. without evidence of blackening. Growth of Enterobacter aerogenes and Escherichia coli is markedly inhibited; colonies References that do grow appear yellow without evidence of blackening. 1. Miller and Tate. 1990. The Maryland Poultryman April:2. Growth of Proteus, Pseudomonas, Providencia, Alteromonas 2. Miller, Tate, Mallinson and Schemer. 1991. Poultry Science 70:2429. 3. Miller, Tate, Mallinson and Schemer. 1992. Poultry Science 71:398. putrefaciens, Yersinia enterocolitica and Acinetobacter 4. Tate, Miller and Mallinson. 1992. J. Food Prot. 55:964. 5. U.S. Department of Agriculture. 1998. Microbiology laboratory guidebook, 3rd ed., Food Safety and calcoaceticus is markedly to completely inhibited on XLT4 Inspection Service, USDA, Washington, D.C. 6. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed. Agar. Shigella species are partially inhibited and colonies ap- American Society for Microbiology, Washington, D.C. 7. Downes and Ito (ed.) 2001. Compendium of methods for the microbiological examination of foods, pear red. 4th ed. American Public Health Association, Washington, D.C. Limitations of the Procedure Availability 1. XLT4 Agar is intended for detecting and isolating Salmonella Difco™ XLT4 Agar Base based on selectivity and colonial characteristics. Presumed USDA Salmonella colonies must be confirmed by biochemical and/ Cat. No. 223420 Dehydrated – 500 g or immunological methods. Consult appropriate references Difco™ XLT4 Agar Supplement for further information.5-7 USDA 2. Non-Salmonella strains that are not completely inhibited Cat. No. 235310 Bottle – l00 mL on this medium may be encountered and must be differen- tiated from Salmonella. Consult appropriate references.5-7

Xanthine Agar (See Nocardia Differentiation Media)

YM Agar • YM Broth

Intended Use ents. Peptone is an additional source of carbon and provides YM Agar and YM Broth are used for cultivating yeasts, molds nitrogen and amino acids. Dextrose provides carbon. Agar is and other aciduric microorganisms. the solidifying agent. Summary and Explanation Formulae YM Agar and YM Broth (Yeast Mold Agar and Broth) are Difco™ YM Agar prepared according to the formulae published by Wickerham.1-3 Approximate Formula* Per Liter Wickerham suggested that YM Broth acidified to pH 3.0-4.0 Yeast Extract ...... 3.0 g Malt Extract...... 3.0 g be used as an enrichment medium for yeasts from populations Peptone ...... 5.0 g also containing bacteria and molds. Dextrose ...... 10.0 g Agar ...... 20.0 g Media selectivity may be enhanced through acidification or Difco™ YM Broth through addition of selective agents. YM Broth may be acidi- Consists of the same ingredients without the agar. fied prior to sterilization. YM Agar should be sterilized with- *Adjusted and/or supplemented as required to meet performance criteria. out pH adjustment and sterile acid added to the sterile molten medium cooled to 45-50°C. Acidified YM Agar should not be Directions for Preparation from heated. Antibiotics may be aseptically added to the sterile me- Dehydrated Product dia. Other fungistatic materials, such as sodium propionate 1. Suspend the powder in 1 L of purified water: ™ and diphenyl may be added to YM Agar to eliminate molds Difco YM Agar – 41 g; ™ and permit the enumeration of yeasts in mixed populations. Difco YM Broth – 21 g. Mix thoroughly. Principles of the Procedure 2. Heat the agar medium with frequent agitation and boil for Yeast extract is a source of trace elements, vitamins and amino 1 minute to completely dissolve the powder. acids. Malt extract is a source of carbon, protein and nutri- 3. Autoclave the agar and broth media at 121°C for 15 minutes.

630